Pasman Yfke, Nagy Eva, Kaushik Azad K
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
Clin Vaccine Immunol. 2012 Aug;19(8):1150-7. doi: 10.1128/CVI.00130-12. Epub 2012 Jun 13.
Single-chain variable antibody fragments (scFvs) with a 2-amino-acid linker capable of multimerization as di-, tri-, or tetrabodies that neutralize bovine herpesvirus type 1 (BoHV-1) in vitro were constructed and expressed in Pichia pastoris. In contrast to the monomeric form, multimeric scFvs had a higher virus neutralization potency, as evidenced by a 2-fold increase in their ability to neutralize BoHV-1 due to avidity effects. Mass spectrum (quadrupole time of flight [Q-TOF]) analyses of multimeric scFv demonstrated extensive heterogeneity due to differential cleavage, variable glycosylation (1 to 9 mannose residues), and the incorporation of minor unidentified adducts. Regardless of the differential glycosylation patterns, the scFvs recognized non-gB or -gE target viral epitopes in the BoHV-1 envelope fraction in a Western blot and also neutralized BoHV-1 in infected Madin-Darby kidney (MDBK) cells in vitro. Indirect evidence for the noncovalent multimerization of scFv was the presence of a major peak of multimerized scFv without a His tag (due to differential cleavage) in the Q-TOF profile, unlike monomeric scFv, which copurified with normally His-tagged scFv and recognized the target antigen. Overall, differentially glycosylated recombinant scFvs against BoHV-1 with a short linker (2 amino acids) are capable of assembly into functional multimers that confer high avidity, resulting in increased virus neutralization in vitro compared to that of monovalent scFv with a long (18-amino-acid) flexible linker. Overall, recombinant multimerized scFv5-2L potentially provides a high-potency therapeutic and immunodiagnostic reagent against BoHV-1, which is suitable for passive immunization and topical application.
构建了具有2个氨基酸连接子、能够多聚化为二聚体、三聚体或四聚体形式单链可变抗体片段（scFv），其能够在体外中和1型牛疱疹病毒（BoHV-1），并在毕赤酵母中表达。与单体形式相比，多聚体scFv具有更高的病毒中和效力，由于亲和力效应，其中和BoHV-1的能力提高了2倍，这证明了这一点。对多聚体scFv的质谱（四极杆飞行时间[Q-TOF]）分析表明，由于差异切割、可变糖基化（1至9个甘露糖残基）以及少量未鉴定加合物的掺入，存在广泛的异质性。无论糖基化模式如何，scFv在蛋白质印迹中识别BoHV-1包膜部分中的非gB或-gE靶病毒表位，并且还能在体外感染的马-达二氏肾（MDBK）细胞中中和BoHV-1。scFv非共价多聚化的间接证据是，在Q-TOF图谱中存在一个没有His标签的多聚化scFv主峰（由于差异切割），这与单体scFv不同，单体scFv与正常带有His标签的scFv共纯化并识别靶抗原。总体而言，具有短连接子（2个氨基酸）的针对BoHV-1的差异糖基化重组scFv能够组装成具有高亲和力的功能性多聚体，与具有长（18个氨基酸）柔性连接子的单价scFv相比，在体外导致病毒中和增加。总体而言，重组多聚化scFv5-2L可能提供一种针对BoHV-1的高效治疗和免疫诊断试剂，适用于被动免疫和局部应用。
